Project Summary/ Abstract Individuals with alcohol use disorder (AUD) show deficits in cognitive function and an inability to regulate ethanol consumption and seeking. These deficits are driven by medial prefrontal cortex (mPFC) dysfunction. Ethanol dependence induced by chronic intermittent ethanol (CIE) vapor exposure disrupts expression of mPFC Kcnc1. This gene encodes voltage-dependent KV3.1 potassium channels, which are highly enriched in parvalbumin positive fast-spiking interneurons (PV+FSIs). Our preliminary data show that CIE-induced dysregulation of mPFC PV+FSI activity is restored by a novel KV3 channel positive modulator which also reduced drinking in dependent and non-dependent mice. These data implicate aberrant KV3 channel activity in PV+FSIs with dependence- induced deficits in mPFC function. We hypothesize that dependence-induced excessive drinking, ethanol reinforcement, and cue-induced relapse-like behavior are regulated by disruption of mPFC KV3 channel activity in PV+FSIs. Specific Aim 1 will examine functional neuroadaptations of KV3 channel activity in mPFC PV+FSIs of G42 (PV-GFP) transgenic mice exposed to CIE or Air, and test the effects of KV3 positive modulators to restore dependence-induced adaptations in PV+FSI function. We will also test if reduced KV3 channel expression is an underlying cellular mechanism driving aberrant KV3 channel activity in PV+FSIs. Specific Aim 2 will test if enhancing KV3 channel activity in the mPFC will reduce ethanol consumption in CIE and Air-exposed mice during home cage drinking sessions. Studies will also determine if KV3 positive modulation can restore mPFC PV+FSI activity during home cage drinking sessions in PV-Cre mice using in vivo fiber photometry techniques. Specific Aim 3 (R00 phase) will shift from studying home cage drinking to studying the role of mPFC KV3 channels in regulating the reinforcing effects of ethanol and relapse-like behavior by using operant conditioning procedures in dependent and non-dependent rats. Using in vivo fiber photometry, studies will test if positive modulation of KV3 increases Ca2+ transients in PV+FSIs in CIE-exposed PV-Cre rats, and in turn, decreases motivation to self-administer ethanol. Experiments will also determine if enhanced KV3 activity reduces ethanol-seeking behavior and modifies Ca2+ transients in PV+FSIs during cue-induced reinstatement tests in CIE-exposed PV-Cre rats. Collectively, these studies will validate KV3 channels as a novel target for the development of effective treatment options for ethanol dependence. In addition, the proposed experiments will provide training in emerging techniques used to selectively measure cellular function in sub-populations of cortical neurons of behaving transgenic animals.